Molecular biosensors capable of signal amplification

ABSTRACT

The present invention provides molecular biosensors capable of signal amplification, and methods of using the molecular biosensors to detect the presence of a target molecule.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/719,867, filed May 22, 2015 which is a continuation of U.S.application Ser. No. 13/578,718, filed Sep. 14, 2012, now issued as U.S.Pat. No. 9,040,287, which is a US National application of PCTApplication PCT/US2011/024547, filed Feb. 11, 2011, which claims thepriority of U.S. provisional application No. 61/303,914, filed Feb. 12,2010, each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to molecular biosensors capable of signalamplification. The biosensors may be used to determine whether a targetmolecule is present in a sample.

BACKGROUND OF THE INVENTION

The detection, identification and quantification of specific moleculesin our environment, food supply, water supply and biological samples(blood, cerebral spinal fluid, urine, et cetera) can be very complex,expensive and time consuming. Methods utilized for detection of thesemolecules include gas chromatography, mass spectroscopy, DNA sequencing,immunoassays, cell-based assays, biomolecular blots and gels, and myriadother multi-step chemical and physical assays.

There continues to be a high demand for convenient methodologies fordetecting and measuring the levels of specific proteins in biologicaland environmental samples. Detecting and measuring levels of proteins isone of the most fundamental and most often performed methodologies inbiomedical research. While antibody-based protein detectionmethodologies are enormously useful in research and medical diagnostics,they are typically not well adapted to rapid, high throughput parallelprotein detection. Hence, there is a need in the art for effective,simple signal amplification and detection means.

BRIEF DESCRIPTION OF THE FIGURES

The application file contains at least one photograph executed in color.Copies of this patent application publication with color photographswill be provided by the Office upon request and payment of the necessaryfee.

FIG. 1 depicts the overall design and function of a two-componentmolecular biosensor comprising a single nicking site.

FIG. 2 depicts the overall design and function of a two-componentmolecular biosensor comprising two nicking sites.

FIG. 3 depicts the overall design and function of a three-componentmolecular biosensor comprising a signaling oligonucleotide attached to abead.

FIG. 4 (A) an agarose gel resolving the digestion products of athree-component molecular biosensor attached to a bead, when increasingconcentrations of the target are added. (B) Quantification results ofdigestion products in (A) using a densitometer.

FIG. 5 depicts FAM signal increase in supernatant when increasingconcentrations of the target are added to a three-component molecularbiosensor attached to a bead.

FIG. 6 depicts anti-HRP ELISA signal increase in supernatant whenincreasing concentrations of the target are added to a three-componentmolecular biosensor attached to a bead. Molecular biosensor, target andrestriction enzyme were added simultaneously.

FIG. 7 depicts FAM signal increase in supernatant with increasingconcentrations of the target when the restriction enzyme is added afterincubation of a target with a three-component molecular biosensorattached to a bead.

FIG. 8 depicts the overall design and function of a three-componentmolecular biosensor comprising a signaling oligonucleotide attached to asolid surface.

FIG. 9 depicts FAM signal increase (A) or HRP ELISA signal increase (B)in supernatant with increasing concentrations of the target using athree-component molecular biosensor attached to a solid surface.

FIG. 10 depicts the overall design and function of a three-componentmolecular biosensor comprising a signaling oligonucleotide not attachedto a solid support.

FIG. 11 depicts FAM signal increase with increasing concentrations ofthe target using a three-component molecular biosensor not attached to asolid support.

FIG. 12 depicts the overall design and function of a three-componentmolecular biosensor comprising a signaling oligonucleotide not attachedto a solid support, using a restriction endonuclease that cleavesoutside the recognition sequence.

FIG. 13 depicts the use of a three-component molecular biosensor fordetection of double-stranded nucleotide sequence binding proteins.

FIG. 14 depicts the use of a three-component molecular biosensor fordetection of ligands of double-stranded nucleotide sequence bindingproteins.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses a molecular biosensor capable ofsignal amplification. Such a biosensor may be used to detect a targetmolecule. In one embodiment, the biosensor is comprised of twocomponents, which comprise two epitope-binding agent constructs.Alternatively, in another embodiment, the biosensor is comprised ofthree components, which comprise two epitope-binding agent constructsand an oligonucleotide construct comprising a restriction enzymerecognition site. Each of these embodiments is discussed in more detailbelow.

Advantageously, a molecular biosensor of the invention, irrespective ofthe embodiment, is capable of signal amplification and provides a rapidhomogeneous means to detect a variety of target molecules, including butnot limited to proteins, carbohydrates, nucleic acids, macromolecules,and analytes.

I. Two-Component Molecular Biosensors

One aspect of the invention encompasses a two-component biosensor andmethods of use thereof. For a two-component biosensor, detection of atarget molecule typically involves target-molecule inducedco-association of two epitope-binding agent constructs (R¹—R²—R³ andR⁴—R⁵—R⁶) that each recognize distinct epitopes on the target molecule.The epitope-binding agent constructs each comprise a single-strandednucleotide sequence (R³ and R⁶). Each single-stranded sequence comprisesa complementary sequence (R⁸ and R⁹). Additionally, at least onesingle-stranded sequence comprises a restriction endonucleaserecognition site (R⁷). Association of the epitope binding agents (R¹ andR⁴) with a target molecule results in annealing of the complementarysequences (R⁸ and R⁹) of the single-stranded nucleotide sequences, suchthat when the complementary regions are extended in the presence of apolymerase, a double-stranded endonuclease recognition site isreconstituted. The newly synthesized double-stranded recognitionsequence may be nicked by a nicking restriction endonuclease thatrecognizes the reconstituted restriction enzyme recognition site. A DNApolymerase may then extend a second nucleic acid from the nick, therebydisplacing the first nicked strand to form a displaced strand. Thesecond extended strand may then be nicked, repeating the extension anddisplacement steps such that multiple copies of the displaced strand areproduced, thereby amplifying the signal from the biosensor. Thedisplaced strand may then be detected via several different methods.

The structure of the biosensor and methods of using the biosensor arediscussed in more detail below.

(a) Biosensor Structure

In exemplary embodiments, a two-component molecular biosensor capable ofsignal amplification comprises two constructs, which together haveformula (I):R¹—R²—R³; andR⁴—R⁵—R⁶;  (I)wherein:

-   -   R¹ is an epitope-binding agent that binds to a first epitope on        a target molecule;    -   R² is a flexible linker attaching R¹ to R³;    -   R³ is a single stranded nucleotide sequence comprising R⁷ and        R⁸;        -   R⁷ is a nucleotide sequence comprising at least one            restriction endonuclease recognition site;        -   R⁸ is a nucleotide sequence complementary to R⁹;    -   R⁶ is a single stranded nucleotide sequence comprising R⁹;        -   R⁹ is a nucleotide sequence complementary to R⁸, such that            when R⁸ and        -   R⁹ associate to form an annealed complex in the presence of            a polymerase, R⁸ and R⁹ are extended by the polymerase to            form a nucleotide sequence complementary to R⁷, forming at            least one double-stranded endonuclease recognition site;    -   R⁵ is a flexible linker attaching R⁴ to R⁶;    -   R⁴ is an epitope-binding agent that binds to a second epitope on        a target molecule.

As will be appreciated by those of skill in the art, the choice ofepitope binding agents, R¹ and R⁴, in molecular biosensors havingformula (I) can and will vary depending upon the particular targetmolecule. By way of example, when the target molecule is a protein, R¹and R⁴ may be an aptamer, or antibody. By way of further example, whenR¹ and R⁴ are double stranded nucleic acid the target molecule istypically a macromolecule that binds to DNA or a DNA binding protein. Ingeneral, suitable choices for R¹ and R⁴ will include two agents thateach recognize distinct epitopes on the same target molecule. In certainembodiments, however, it is also envisioned that R¹ and R⁴ may recognizedistinct epitopes on different target molecules. Non-limiting examplesof suitable epitope binding agents may include agents selected from thegroup consisting of an aptamer, an antibody, an antibody fragment, adouble-stranded DNA sequence, modified nucleic acids, nucleic acidmimics (e.g. LNA or PNA), a ligand, a ligand fragment, a receptor, areceptor fragment, a polypeptide, a peptide, a coenzyme, a coregulator,an allosteric molecule, a chemical entity and an ion.

In one embodiment, R¹ and R⁴ are each aptamers having a sequence rangingin length from about 20 to about 110 bases. In another embodiment, R¹and R⁴ are each antibodies or antibody-like binders selected from thegroup consisting of polyclonal antibodies, ascites, Fab fragments, Fab′fragments, monoclonal antibodies, humanized antibodies, chimericantibodies, single chain antibodies, and non-immunoglobulin scaffoldssuch as Affibodies, Anticalins, designed Ankyrin repeat proteins andothers. In an alternative embodiment, R¹ and R⁴ are peptides. In anexemplary embodiment, R¹ and R⁴ are each monoclonal antibodies. In anadditional embodiment, R¹ and R⁴ are each double stranded DNA. In afurther embodiment, R¹ is a double stranded nucleic acid and R⁴ is anaptamer. In an additional embodiment, R¹ is an antibody and R⁴ is anaptamer. In another additional embodiment, R¹ is an antibody and R⁴ is adouble stranded DNA.

In an additional embodiment for molecular biosensors having formula (I),exemplary linkers, R² and R⁵, will functionally keep R³ and R⁶ in closeproximity such that when R¹ and R⁴ each bind to the target molecule, R⁸and R⁹ associate in a manner such that a detectable signal is produced.R² and R⁵ may each be a nucleotide sequence from about 10 to about 100nucleotides in length. In one embodiment, R² and R⁵ are from 10 to about25 nucleotides in length. In another embodiment, R² and R⁵ are fromabout 25 to about 50 nucleotides in length. In a further embodiment, R²and R⁵ are from about 50 to about 75 nucleotides in length. In yetanother embodiment, R² and R⁵ are from about 75 to about 100 nucleotidesin length. In each embodiment, the nucleotides comprising the linkersmay be any of the nucleotide bases in DNA or RNA (A, C, T, G in the caseof DNA, or A, C, U, G in the case of RNA). In one embodiment R² and R⁵are comprised of DNA bases. In another embodiment, R² and R⁵ arecomprised of RNA bases. In yet another embodiment, R² and R⁵ arecomprised of modified nucleic acid bases, such as modified DNA bases ormodified RNA bases. Modifications may occur at, but are not restrictedto, the sugar 2′ position, the C-5 position of pyrimidines, and the8-position of purines. Examples of suitable modified DNA or RNA basesmay include 2′-fluoro nucleotides, 2′-amino nucleotides,5′-aminoallyl-2′-fluoro nucleotides and phosphorothioate nucleotides(monothiophosphate and dithiophosphate). In a further embodiment, R² andR⁵ may be nucleotide mimics. Examples of nucleotide mimics may includelocked nucleic acids (LNA), peptide nucleic acids (PNA), andphosphorodiamidate morpholine oligomers (PMO). Alternatively, R² and R⁵may be a bifunctional chemical linker, or a polymer of bifunctionalchemical linkers. In one embodiment the bifunctional chemical linker isheterobifunctional. Suitable heterobifunctional chemical linkers mayinclude sulfoSMCC (sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), and Ic-SPDP(N-succinimidyl-6-(3′-(2-pyridyldithio)-propionamido)-hexanoate). Inanother embodiment the bifunctional chemical linker is homobifunctional.Suitable homobifunctional linkers may include disuccinimidyl suberate,disuccinimidyl glutarate, and disuccinimidyl tartrate. Additionalsuitable linkers may include the phosphoramidate form of Spacer 18comprised of polyethylene glycol. In one embodiment, R² and R⁵ are from0 to about 500 angstroms in length. In another embodiment, R² and R⁵ arefrom about 20 to about 400 angstroms in length. In yet anotherembodiment, R² and R⁵ are from about 50 to about 250 angstroms inlength.

In a further embodiment for molecular biosensors having formula (I), R³comprises R⁷ and R⁸, and R⁶ comprises R⁹. Generally speaking, except forR⁸ and R⁹, R³ and R⁶ are not complementary. Wand R⁹ are nucleotidesequences that are complementary to each other such that they preferablydo not associate unless R¹ and R⁴ bind to separate epitopes on a targetmolecule. When R¹ and R⁴ bind to separate epitopes of a target molecule,R⁸ and R⁹ are brought into relative proximity resulting in an increasein their local concentration, which drives the association of R⁸ and R⁹.

To ensure that R⁸ and R⁹ only associate when R¹ and R⁴ bind to separateepitopes of a target, R⁸ and R⁹ generally have a length such that thefree energy of association is from about −5 to about −12 kcal/mole at atemperature from about 21° C. to about 40° C. and at a saltconcentration from about 1 mM to about 100 mM. In other embodiments, thefree energy of association between R⁸ and R⁹ is about −5 kcal/mole,about −6 kcal/mole, about −7 kcal/mole, about −8 kcal/mole, about −9kcal/mole, about −10 kcal/mole, about −11 kcal/mole, or greater thanabout −12 kcal/mole at a temperature from about 21° C. to about 40° C.and at a salt concentration from about 1 mM to about 100 mM. Inadditional embodiments, R⁸ and R⁹ may range from about 4 to about 20nucleotides in length. In other embodiments, R⁸ and R⁹ may be about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 11, about12, about 13, about 14, about 15, about 16, about 17, about 18, about19, or greater than about 10 nucleotides in length.

In some embodiments, R³ comprises R⁷—R⁸, such that R⁷ is located 5′ toR⁸. In other embodiments, R³ comprises R⁸—R⁷, such that R⁸ is located 5′to R⁷.

In an exemplary embodiment, R⁸ and R⁹ are at the 3′ ends of R³ and R⁶,such that association of R⁸ and R⁹ forms a complex where the 3′ ends canbe extended using R³ and R⁶ as a template to form a double-strandednucleotide sequence comprising R⁷. Polymerases suitable for extending R⁸and R⁹ are known in the art. For example, non-limiting examples ofnucleotide polymerases suitable for extending nucleic acid sequences ofthe invention may include Bsu DNA Polymerase, DNA Polymerase I (E.coli), DNA Polymerase I Large (Klenow) Fragment, Klenow Fragment (3″→5′exo-), phi29 DNA Polymerase, T4 DNA Polymerase, T7 DNA Polymerase(unmodified), or any of the thermophilic polymerases, such as the fulllength or large fragment of Bst DNA Polymerase, Taq DNA Polymerase, 9°N_(m) DNA Polymerase, Crimson Taq DNA Polymerase, Deep VentR™ (exo-) DNAPolymerase, Deep VentR™ DNA Polymerase, DyNAzyme™ EXT DNA Polymerase,DyNAzyme™ II Hot Start DNA Polymerase, Hemo KlenTaq™, Phusion®High-Fidelity DNA Polymerase, Sulfolobus DNA Polymerase IV, Therminator™DNA Polymerase, VentR® DNA Polymerase.

Generally speaking, for molecular biosensors having formula (I) R³comprises at least one restriction endonuclease recognition site. Insome embodiments, however, R³ may comprise more than one restrictionendonuclease recognition site. For instance, R³ may comprise at leasttwo, three, four, or five endonuclease recognition sites. Similarly, R⁶may comprise at least one, two, three, four or five endonucleaserecognition sites.

Typically, a restriction enzyme recognizing a restriction enzymerecognition site cannot cleave or nick a single stranded nucleotidesequence. Association of the epitope binding agents with a targetmolecule and the subsequent extension of the 3′ ends of R⁸ and R⁹ in thepresence of a polymerase forms a double-stranded endonucleaserecognition site that may be cleaved or nicked by a restrictionendonuclease. As is commonly known by persons skilled in the art,restriction endonucleases may hydrolyze both strands of the nucleic acidduplex to cleave the nucleic acid duplex, or hydrolyze one of thestrands of the nucleic acid duplex, thus producing double-strandednucleic acid molecules that are “nicked”, rather than cleaved. Inpreferred embodiments of molecular biosensors having formula (I), R⁷comprises an endonuclease recognition sequence for a nicking restrictionenzyme. A nicking restriction endonuclease may hydrolyze the bottom orthe top strand of a nucleic acid duplex. By way of non-limiting example,recognition sites for nicking restriction enzymes may include Nt.BstNBI,Nb.BsrD, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvC and Nb.BsmI.

In each of the foregoing embodiments for molecular biosensors havingformula (I), the first nucleic acid construct, R¹—R²—R³ and the secondnucleic acid construct, R⁴—R⁵—R⁶, may optionally be attached to eachother by a linker R^(LA) to create tight binding bivalent ligands.Typically, the attachment is by covalent bond formation. Alternatively,the attachment may be by non covalent bond formation. In one embodiment,R^(LA) attaches R¹ of the first nucleic acid construct to R⁴ of thesecond nucleic acid construct to form a molecule comprising:

In a further embodiment, R^(LA) attaches R² of the first nucleic acidconstruct to R5 of the second nucleic acid construct to form a moleculecomprising:

In yet another embodiment, R^(LA) attaches R³ of the first nucleic acidconstruct to R⁷ of the second nucleic acid construct to form a moleculecomprising:

Generally speaking, R^(LA) may be a nucleotide sequence from about 10 toabout 100 nucleotides in length. The nucleotides comprising R^(LA) maybe any of the nucleotide bases in DNA or RNA (A, C, T, G in the case ofDNA, or A, C, U, G in the case of RNA). In one embodiment, R^(LA) iscomprised of DNA bases. In another embodiment, R^(LA) is comprised ofRNA bases. In yet another embodiment, R^(LA) is comprised of modifiednucleic acid bases, such as modified DNA bases or modified RNA bases.Modifications may occur at, but are not restricted to, the sugar 2′position, the C-5 position of pyrimidines, and the 8-position ofpurines. Examples of suitable modified DNA or RNA bases may include2′-fluoro nucleotides, 2′-amino nucleotides, 5′-aminoallyl-2′-fluoronucleotides and phosphorothioate nucleotides (monothiophosphate anddithiophosphate). In a further embodiment, R^(LA) is comprised ofnucleotide mimics. Examples of nucleotide mimics may include lockednucleic acids (LNA), peptide nucleic acids (PNA), and phosphorodiamidatemorpholine oligomers (PMO). Alternatively, R^(LA) may be a bifunctionalchemical linker or a polymer of bifunctional chemical linkers. In oneembodiment the bifunctional chemical linker is heterobifunctional.Suitable heterobifunctional chemical linkers may include sulfoSMCC(Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), andIc-SPDP(N-Succinimidyl-6-(3′-(2-PyridylDithio)-Propionamido)-hexanoate). Inanother embodiment, the bifunctional chemical linker ishomobifunctional. Suitable homobifunctional linkers may includedisuccinimidyl suberate, disuccinimidyl glutarate, and disuccinimidyltartrate. An exemplary R^(LA) is the phosphoramidate form of Spacer 18comprised of polyethylene glycol. In one embodiment, R^(LA) is fromabout 1 to about 500 angstroms in length. In another embodiment, R^(LA)is from about 20 to about 400 angstroms in length. In yet anotherembodiment, R^(LA) is from about 50 to about 250 angstroms in length.

(b) Means of Detection

As discussed above, when R⁸ and R⁹ are extended in the presence of apolymerase, the newly synthesized double-stranded endonucleaserecognition sequence may be nicked by a nicking restriction endonucleasethat recognizes the double-stranded restriction enzyme recognition site.A DNA polymerase may then extend a second nucleic acid from the nick,thereby displacing the first nicked strand to form a displaced strand.The second extended strand may then be nicked, repeating the extensionand displacement steps such that multiple copies of the displaced strandare produced, thereby amplifying the signal from the biosensor. Thedisplaced strand may then be detected via several different methods.Three such methods are detailed below.

i. Double-Stranded Nucleic Acid Stains

In some embodiments, a displaced strand may be detected and/orquantitated by contacting a displaced strand with a complementarynucleic acid sequence. The resulting double-stranded nucleotide sequencemay be detected using nucleic acid staining methods specific fordouble-stranded sequences. Non-limiting examples of nucleic acid stainsthat may be used for detecting double-stranded nucleotide sequences mayinclude ethidium bromide, thiazole orange, propidium iodide, DAPI,Hoechst dyes, acridine orange, 7-AAD, LDS 751, hydroxystilbamidine, andcyanine dyes such as TOTO-1, POPO-1, BOBO-1, YOYO-1, JOJO-1, LOLO-1,POPO-3, YOYO-3, TOTO-3, BOBO-3, PicoGreen, SYBR Gold, SYBR Green I andSYBR Green II.

ii. Type IIS Endonuclease Construct

In another embodiment, a displaced strand may be detected and/orquantitated by associating with a Type IIS endonuclease nucleic acidconstruct. The nucleic acid construct may generally comprise twostrands, where the first strand comprises R¹⁰—R¹²—R¹⁴ and the secondstrand comprises R¹¹—R¹³. R¹⁴ is complementary to the displaced strand,and when associated with a displaced strand, comprises a Type IISendonuclease recognition site. R¹² is complementary to R¹³, andtogether, R¹² and R¹³ comprise a cleavage site for a Type IISendonuclease. R¹² and R¹³ are of such a length that the two strands(i.e. R¹⁰—R¹²—R¹⁴ and R¹¹—R¹³) stay hybridized in the absence of thedisplaced strand. R¹⁰ and R¹¹ comprise a detection means, such that whenR¹² and R¹³ are cleaved by a Type IIS endonuclease, R¹⁰ and R¹¹ arereleased from the Type IIS endonuclease construct and produce adetectable signal. Suitable detection means for R¹⁰ and R¹¹ may comprisefluorescent resonance energy transfer (FRET), lanthamide resonanceenergy transfer (LRET), fluorescence cross-correlation spectroscopy,fluorescence quenching, fluorescence polarization, flow cytometry,scintillation proximity, luminescence resonance energy transfer, directquenching, ground-state complex formation, chemiluminescence energytransfer, bioluminescence resonance energy transfer, excimer formation,colorimetric substrates detection, phosphorescence, electrochemicalchanges, and redox potential changes. (See FIG. 1 E2.)

iii. Linker Construct

In some embodiments, a displaced strand may be detected by a linkerconstruct. Usually, a linker construct comprisesR¹⁵—R¹⁶—R¹⁷—R¹⁸—R¹⁹—R²⁰—R²¹. R¹⁸ is a nucleotide sequence that iscomplementary to the displaced strand, and together with the displacedstrand, comprises an endonuclease recognition site. R¹⁷ and R¹⁹ arelinkers, and may be defined as R² and R⁵ above. R¹⁶ and R²⁰ arecomplementary nucleic acid sequences, and may be defined as R⁸ and R⁹above. R¹⁵ and R²¹ comprise a detection means, and may be defined as R¹⁰and R¹¹ above. (See FIG. 1 E3)

When R¹⁸ binds to a displaced strand, a double-stranded restrictionendonuclease recognition site is formed. In the presence of arestriction endonuclease, R¹⁸ and the displaced strand are cleaved atthe endonuclease recognition site. This destabilizes the association ofR¹⁶ and R²⁰, resulting in the separation of R¹⁵ and R²¹. This separationresults in a detectable and quantifiable change in signal intensity.

II. Three-Component Molecular Biosensors

Another aspect of the invention encompasses a three-component biosensorcapable of signal amplification. In a three-component embodiment,analogous to a two-component sensor, detection of a target moleculetypically involves target-molecule induced co-association of twoepitope-binding agent constructs that each recognize distinct epitopeson the target molecule. Unlike the two-component embodiment, however,the epitope-binding agent constructs each comprise single strandednucleic acid sequences that are complementary to two distinct regions ofthe oligonucleotide construct, as opposed to being complementary to eachother (as in the two-component sensor). Co-association of the twoepitope-binding agent constructs with a target molecule results inhybridization of each single stranded nucleic acid sequence to theoligonucleotide construct. This tripartite construct comprised of thetwo single stranded nucleic acid sequences and the oligonucleotideconstruct reconstitutes a restriction endonuclease recognition site. Theendonuclease recognition site may be cleaved in the presence of arestriction endonuclease. Such cleavage destabilizes the association ofthe single stranded nucleic acid sequences and the (now cleaved)oligonucleotide construct, releasing the single stranded nucleic acidsequences. The single stranded nucleic acid sequences may then bind toanother oligonucleotide construct, repeating the cleavage cycle andtherefore amplifying the biosensor signal. Importantly, theoligonucleotide construct is capable of producing a detectable signalwhen cleaved.

In certain embodiments, the three-component molecular biosensor willcomprise a solid support. In alternative embodiments, thethree-component molecular biosensor will not comprise a solid support.Both of these embodiments are discussed in more detail below. In someembodiments, a three-component molecular biosensor may comprise aplurality of oligonucleotide constructs (e.g. R⁷—R⁸ or R⁷—R⁸—R⁹).

(a) Three Component Molecular Biosensors Comprising a Solid Support

In one embodiment, a three-component molecular biosensor will comprisean oligonucleotide construct attached to a solid support. Generallyspeaking, co-association of the two epitope-binding agent constructswith a target molecule results in hybridization of each single strandednucleic acid sequence to the oligonucleotide construct, producing atripartite double-stranded nucleic acid molecule that contains arestriction endonuclease recognition site. In the presence of arestriction endonuclease, the oligonucleotide construct may be cleavedto release a signaling molecule from the solid support. (See, forinstance, FIG. 3)

For example, in some embodiments the three-component molecular biosensorcomprises at least three constructs, which together have formula (II):R¹—R²—R³;R⁴—R⁵—R⁶; andat least one R⁷—R⁸—R⁹;  (II)wherein:

-   -   R¹ is an epitope-binding agent that binds to a first epitope on        a target molecule;    -   R² is a flexible linker attaching R¹ to R³;    -   R³ and R⁶ are a first pair of nucleotide sequences that are        complementary to two distinct regions on R⁸;    -   R⁵ is a flexible linker attaching R⁴ to R⁶;    -   R⁴ is an epitope-binding agent that binds to a second epitope on        a target molecule;    -   R⁸ is a nucleotide construct comprising a first region that is        complementary to R³ and a second region that is complementary to        R⁶, such that when R³ and R⁶ associated with R⁸, an endonuclease        restriction site is reconstituted;    -   R⁷ is a signaling molecule; and    -   R⁹ is a solid support.

The choice of epitope binding agents, R¹ and R⁴, in molecular biosensorshaving formula (II) can and will vary depending upon the particulartarget molecule. By way of example, when the target molecule is aprotein, R¹ and R⁴ may be an aptamer, or antibody. By way of furtherexample, when R¹ and R⁴ are double stranded nucleic acid the targetmolecule is typically a macromolecule that binds to DNA or a DNA bindingprotein. In general, suitable choices for R¹ and R⁴ will include twoagents that each recognize distinct epitopes on the same targetmolecule. In certain embodiments, however, it is also envisioned that R¹and R⁴ may recognize distinct epitopes on different target molecules.Non-limiting examples of suitable epitope binding agents, depending uponthe target molecule, may include agents selected from the groupconsisting of an aptamer, an antibody, an antibody fragment, adouble-stranded DNA sequence, modified nucleic acids, nucleic acidmimics, a ligand, a ligand fragment, a receptor, a receptor fragment, apolypeptide, a peptide, a coenzyme, a coregulator, an allostericmolecule, and an ion. In an exemplary embodiment, R¹ and R⁴ are eachaptamers having a sequence ranging in length from about 20 to about 110bases. In another embodiment, R¹ and R⁴ are each antibodies selectedfrom the group consisting of polyclonal antibodies, ascites, Fabfragments, Fab′ fragments, monoclonal antibodies, humanized antibodies,chimeric antibodies, and single-chain antibodies. In an alternativeembodiment, R¹ and R⁴ are peptides. In a preferred embodiment, R¹ and R⁴are each monoclonal antibodies. In an additional embodiment, R¹ and R⁴are each double stranded DNA. In a further embodiment, R¹ is a doublestranded nucleic acid and R⁴ is all aptamer. In an additionalembodiment, R¹ is an antibody and R⁴ is an aptamer. In anotheradditional embodiment, R¹ is an antibody and R⁴ is a double strandedDNA.

In an additional embodiment for molecular biosensors having formula(II), exemplary linkers, R² and R⁵, will functionally keep R³ and R⁶ inappropriate proximity such that when R¹ and R⁴ each bind to the targetmolecule, R³ and R⁶ associate with R⁸ producing a detectable signal. R²and R⁵ may each be a nucleotide sequence from about 10 to about 100nucleotides in length. In one embodiment, R² and R⁵ are from about 10 toabout 25 nucleotides in length. In another embodiment, R² and R⁵ arefrom about 25 to about 50 nucleotides in length. In a furtherembodiment, R² and R⁵ are from about 50 to about 75 nucleotides inlength. In yet another embodiment, R² and R⁵ are from about 75 to about100 nucleotides in length. In each embodiment, the nucleotidescomprising the linkers may be any of the nucleotide bases in DNA or RNA(A, C, T, G in the case of DNA, or A, C, U, G in the case of RNA). Inone embodiment R² and R⁵ are comprised of DNA bases. In anotherembodiment, R² and R⁵ are comprised of RNA bases. In yet anotherembodiment, R² and R⁵ are comprised of modified nucleic acid bases, suchas modified DNA bases or modified RNA bases. Modifications may occur at,but are not restricted to, the sugar 2′ position, the C-5 position ofpyrimidines, and the 8-position of purines. Examples of suitablemodified DNA or RNA bases may include 2′-fluoro nucleotides, 2′-aminonucleotides, 5′-aminoallyl-2′-fluoro nucleotides and phosphorothioatenucleotides (monothiophosphate and dithiophosphate). In a furtherembodiment, R² and R⁵ may be nucleotide mimics. Examples of nucleotidemimics may include locked nucleic acids (LNA), peptide nucleic acids(PNA), and phosphorodiamidate morpholine oligomers (PMO).

Alternatively, R² and R⁵ may be a bifunctional chemical linker or apolymer of bifunctional chemical linkers. In one embodiment thebifunctional chemical linker is heterobifunctional. Suitableheterobifunctional chemical linkers may include sulfoSMCC(Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate), andIc-SPDP(N-Succinimidyl-6-(3′-(2-PyridylDithio)-Propionamido)-hexanoate). Inanother embodiment the bifunctional chemical linker is homobifunctional.Suitable homobifunctional linkers may include disuccinimidyl suberate,disuccinimidyl glutarate, and disuccinimidyl tartrate. Additionalsuitable linkers may include the phosphoramidate form of Spacer 18comprised of polyethylene glycol. In one embodiment, R² and R⁵ are from0 to about 500 angstroms in length. In another embodiment, R² and R⁵ arefrom about 20 to about 400 angstroms in length. In yet anotherembodiment, R² and R⁵ are from about 50 to about 250 angstroms inlength.

R⁷ of formula (II) is a signaling molecule. Suitable signaling moleculesare known in the art. Non-limiting examples may include luminescentmolecules, chemiluminescent molecules, fluorochromes, fluorescentquenching agents, colored molecules, radioisotopes, scintillants,massive labels (for detection via mass changes), biotin, avidin,streptavidin, protein A, protein G, antibodies or fragments thereof,Grb2, polyhistidine, Ni²⁺, Flag tags, myc tags, heavy metals, enzymes,alkaline phosphatase, peroxidase, luciferase, electron donors/acceptors,acridinium esters, and colorimetric substrates. The skilled artisanwould readily recognize other useful labels that are not mentionedabove, which may be employed in the operation of the present invention.

For molecular biosensors having formula (II), R⁸ comprises a firstregion that is complementary to R⁶, and a second region that iscomplementary to R³. R⁸ may be from about 8 to about 100 nucleotides inlength. In other embodiments, R⁸ is from about 10 to about 15nucleotides in length, or from about 15 to about 20 nucleotides inlength, or from about 20 to about 25 nucleotides in length, or fromabout 25 to about 30 nucleotides in length, or from about 30 to about 35nucleotides in length, or from about 35 to about 40 nucleotides inlength, or from about 40 to about 45 nucleotides in length, or fromabout 45 to about 50 nucleotides in length, or from about 50 to about 55nucleotides in length, or from about 55 to about 60 nucleotides inlength, or from about 60 to about 65 nucleotides in length, or fromabout 65 to about 70 nucleotides in length, or from about 70 to about 75nucleotides in length, or from about 75 to about 80 nucleotides inlength, or from about 80 to about 85 nucleotides in length, or fromabout 85 to about 90 nucleotides in length, or from about 90 to about 95nucleotides in length, or greater than about 95 nucleotides in length.

When R³ and R⁶ associate with R⁸, a tripartite double-stranded DNAmolecule is formed that contains a restriction endonuclease recognitionsequence. In the presence of a restriction endonuclease, R⁸ is cleaved,releasing R⁷ from the solid support R⁹. In an exemplary embodiment, R³and R⁶ do not form a stable complex with R⁸ after R⁸ is cleaved, freeingR³ and R⁶ to bind to another R⁸ and repeat the cleavage cycle. Thisamplifies the biosensor signal.

In an exemplary embodiment, R⁸ will comprise formula (III):R¹⁰—R¹¹—R¹²—R¹³  (III)wherein:

-   -   R¹⁰ and R¹³ are single-stranded nucleotide sequences not        complementary to any of R¹, R², R³, R⁴, R⁵, or R⁶;    -   R¹¹ is a nucleotide sequence complementary to R³; and    -   R¹² is a nucleotide sequence that is complementary to R⁶.

In some embodiments, R¹⁰ and R¹³ may independently be from about 0 toabout 20 nucleotides in length. In other embodiments, R¹⁰ and R¹³ mayindependently be from about 2 to about 4 nucleotides in length, or fromabout 4 to about 6 nucleotides in length, or from about 6 to about 8nucleotides in length, or from about 8 to about 10 nucleotides inlength, or from about 10 to about 12 nucleotides in length, or fromabout 12 to about 14 nucleotides in length, or from about 14 to about 16nucleotides in length, or from about 16 to about 18 nucleotides inlength, or from about 18 to about 20 nucleotides in length, or greaterthan about 20 nucleotides in length.

Generally speaking, R¹¹ and R¹² have a length such that the free energyof association between R¹¹ and R³ and R¹² and R⁶ is from about −5 toabout −12 kcal/mole at a temperature from about 21° C. to about 40° C.and at a salt concentration from about 1 mM to about 100 mM. In otherembodiments, the free energy of association between R¹¹ and R³ and R¹²and R⁶ is about −5 kcal/mole, about −6 kcal/mole, about −7 kcal/mole,about −8 kcal/mole, about −9 kcal/mole, about −10 kcal/mole, about −11kcal/mole, or greater than about −12 kcal/mole at a temperature fromabout 21° C. to about 40° C. and at a salt concentration from about 1 mMto about 100 mM. In additional embodiments, R¹¹ and R¹² may range fromabout 4 to about 20 nucleotides in length. In other embodiments, R¹¹ andR¹² may be about 4, about 5, about 6, about 7, about 8, about 9, about10, about 11, about 12, about 13, about 14, about 15, about 16, about17, about 18, about 19, or greater than about 10 nucleotides in length.

In one embodiment, when R⁸ comprises formula (III), the cleavage site ofthe restriction endonuclease recognition sequence produced by theassociation of R³ and R⁶ with R⁸ is located between R¹¹ and R¹². In thismanner, in the presence of a suitable restriction endonuclease, R⁸ willbe cleaved between R¹¹ and R¹², but R³ and R⁶ remain intact. Suitablerestriction endonuclease recognition sequences are recognized byrestriction enzymes that cleave double stranded nucleic acid, but notsingle stranded nucleic acid. Such enzymes and the correspondingrecognition sites are known in the art. By way of non-limiting example,these enzymes may include AccI, AgeI, BamHI, BglI, BgIII, BsiWI, BstBI,ClaI, CviQI, DdeI, DpnI, DraI, EagI, EcoRI, EcoRV, FseI, FspI, HaelI,HaeII, HhaI, HincII, HinDIII, HpaI, HpaII, KpnI, KspI, MboI, MfeI, NaeI,NarI, NcoI, NdeI, NheI, NotI, PhoI, PstI, PvuI, PvulI, SacI, SacI, SalI,SbfI, SmaI, SpeI, SphI, StuI, TaqI, TliI, TfiI, XbaI, XhoI, XmaI, XmnI,and ZraI.

In another exemplary embodiment, R⁸ will comprise formula (IV):R¹⁰—R¹¹—R¹²—R¹³—R¹⁴—R¹⁵  (IV)wherein:

-   -   R¹¹, R¹², R¹³, and R¹⁴ are single stranded oligonucleotide        sequences not complementary to each other or any of R¹, R², R³,        R⁴, R⁵, or R⁶;    -   R¹⁰ and R¹⁵ are double-stranded nucleic acid sequences;    -   R¹² is a nucleotide sequence complementary to R³; and    -   R¹³ is a nucleotide sequence that is complementary to R⁶.

R¹¹ and R¹⁴ may independently be from about 0 to about 20 nucleotides inlength. In other embodiments, R¹¹ and R¹⁴ may independently be fromabout 2 to about 4 nucleotides in length, or from about 4 to about 6nucleotides in length, or from about 6 to about 8 nucleotides in length,or from about 8 to about 10 nucleotides in length, or from about 10 toabout 12 nucleotides in length, or from about 12 to about 14 nucleotidesin length, or from about 14 to about 16 nucleotides in length, or fromabout 16 to about 18 nucleotides in length, or from about 18 to about 20nucleotides in length, or greater than about 20 nucleotides in length;

R¹⁰ and R¹⁵ may independently be from about 0 to about 20 base pairs inlength. In other embodiments, R¹⁰ and R¹⁵ may independently be fromabout 2 to about 4 base pairs in length, or from about 4 to about 6 basepairs in length, or from about 6 to about 8 base pairs in length, orfrom about 8 to about 10 base pairs in length, or from about 10 to about12 base pairs in length, or from about 12 to about 14 base pairs inlength, or from about 14 to about 16 base pairs in length, or from about16 to about 18 base pairs in length, or from about 18 to about 20 basepairs in length, or greater than about 20 base pairs in length;

R¹² and R¹³ generally have a length such that the free energy ofassociation between R¹² and R³ and R¹³ and R⁶ is from about −5 to about−12 kcal/mole at a temperature from about 21° C. to about 40° C. and ata salt concentration from about 1 mM to about 100 mM. In otherembodiments, the free energy of association between R¹² and R³ and R¹³and R⁶ is about −5 kcal/mole, about −6 kcal/mole, about −7 kcal/mole,about −8 kcal/mole, about −9 kcal/mole, about −10 kcal/mole, about −11kcal/mole, or greater than about −12 kcal/mole at a temperature fromabout 21° C. to about 40° C. and at a salt concentration from about 1 mMto about 100 mM. In additional embodiments, R¹² and R¹³ may range fromabout 4 to about 20 nucleotides in length. In other embodiments, R¹² andR¹³ may be about 4, about 5, about 6, about 7, about 8, about 9, about10, about 11, about 12, about 13, about 14, about 15, about 16, about17, about 18, about 19, or greater than about 20 nucleotides in length.

In yet another exemplary embodiment, R⁸ may comprise formula (V):R¹⁰—R¹¹—R¹²—R¹³—R¹⁴—R¹⁵—R¹⁶  (V)wherein:

R¹¹, R¹², R¹⁴, R¹⁵ and R¹⁶ are single stranded oligonucleotide sequencesindependently not complementary to each other or any of R¹, R², R³, R⁴,R⁵, or R⁶;

-   -   R¹⁰ and R¹³ are double-stranded nucleic acid sequences;    -   R¹¹ is a nucleotide sequence complementary to R³; and    -   R¹⁵ is a nucleotide sequence that is complementary to R⁶.

R¹², R¹⁴, and R¹⁶ may independently be from about 0 to about 20nucleotides in length. In other embodiments, R¹², R¹⁴, and R¹⁶ mayindependently be from about 2 to about 4 nucleotides in length, or fromabout 4 to about 6 nucleotides in length, or from about 6 to about 8nucleotides in length, or from about 8 to about 10 nucleotides inlength, or from about 10 to about 12 nucleotides in length, or fromabout 12 to about 14 nucleotides in length, or from about 14 to about 16nucleotides in length, or from about 16 to about 18 nucleotides inlength, or from about 18 to about 20 nucleotides in length, or greaterthan about 20 nucleotides in length.

R¹⁰ and R¹³ may independently be from about 0 to about 20 base pairs inlength. In other embodiments, R¹⁰ and R¹³ may independently be fromabout 2 to about 4 base pairs in length, or from about 4 to about 6 basepairs in length, or from about 6 to about 8 base pairs in length, orfrom about 8 to about 10 base pairs in length, or from about 10 to about12 base pairs in length, or from about 12 to about 14 base pairs inlength, or from about 14 to about 16 base pairs in length, or from about16 to about 18 base pairs in length, or from about 18 to about 20 basepairs in length, or greater than about 20 base pairs in length.

R¹¹ and R¹⁵ generally have a length such that the free energy ofassociation between R¹¹ and R³ and R¹⁵ and R⁶ is from about −5 to about−12 kcal/mole at a temperature from about 21° C. to about 40° C. and ata salt concentration from about 1 mM to about 100 mM. In otherembodiments, the free energy of association between R¹¹ and R³ and R¹⁵and R⁶ is about −5 kcal/mole, about −6 kcal/mole, about −7 kcal/mole,about −8 kcal/mole, about −9 kcal/mole, about −10 kcal/mole, about −11kcal/mole, or greater than about −12 kcal/mole at a temperature fromabout 21° C. to about 40° C. and at a salt concentration from about 1 mMto about 100 mM. In additional embodiments, R¹¹ and R¹⁵ may range fromabout 4 to about 20 nucleotides in length. In other embodiments, R¹¹ andR¹⁵ may be about 4, about 5, about 6, about 7, about 8, about 9, about10, about 11, about 12, about 13, about 14, about 15, about 16, about17, about 18, about 19, or greater than about 10 nucleotides in length.

When R⁸ comprises formula (IV) or formula (V), a cleavage site of arestriction endonuclease recognition sequence produced by theassociation of R³ and R⁶ with R⁸ may be located within R¹⁰ for eitherformula (IV) or formula (V), R¹⁵ for formula (IV), R¹³ for formula (V),or a combination thereof. Suitable restriction endonuclease recognitionsequences for these embodiments are recognized by restriction enzymesthat cleave double stranded nucleic acid outside the recognitionsequence of the restriction enzyme. Such enzymes and the correspondingrecognition and cleavage sites are known in the art. By way ofnon-limiting example, these sites may include AcuI, AlwI, BaeI, BbsI,BbvI, BccI, BceAI, BcgI, BciVI, BfuAI, BmrI, BpmI, BpuEI, BsaI, BsaXI,BseRI, BsgI, BsmAI, BsmBI, BsmFI, BspCNI, BspMI, BspQI, BtgZI, CspCI,EarI, EciI, EcoP15I, FokI, HgaI, HphI, HpyAV, MboII, MlyI, MmeI,MmeAIII, PleI, SapI, SfaNI.

In some embodiments for molecular biosensors having Formula (IV) orFormula (V), R⁷ may comprise two signaling molecules, each attached toone strand of a double-stranded nucleotide sequence comprising R⁸.Cleavage of the restriction enzyme recognition site results in therelease and separation of the two signaling molecules, resulting in adetectable and quantifiable change in signal intensity. Exemplarydetections means suitable for use in the molecular biosensors includefluorescent resonance energy transfer (FRET), lanthamide resonanceenergy transfer (LRET), fluorescence cross-correlation spectroscopy,fluorescence quenching, fluorescence polarization, flow cytometry,scintillation proximity, luminescence resonance energy transfer, directquenching, ground-state complex formation, chemiluminescence energytransfer, bioluminescence resonance energy transfer, excimer formation,colorimetric substrates detection, phosphorescence, electrochemicalchanges, and redox potential changes.

In some embodiments, R⁹ is a solid support having R⁸ attached thereto.Non-limiting examples of suitable solid supports may include microtitreplates, test tubes, beads, resins and other polymers, as well as othersurfaces either known in the art or described herein. The solid supportmay be a material that may be modified to contain discrete individualsites appropriate for the attachment or association of the construct andis amenable to at least one detection method. Non-limiting examples ofsolid support materials include glass, modified or functionalized glass,plastics (including acrylics, polystyrene and copolymers of styrene andother materials, polypropylene, polyethylene, polybutylene,polyurethanes, TeflonJ, etc.), nylon or nitrocellulose, polysaccharides,nylon, resins, silica or silica-based materials including silicon andmodified silicon, carbon, metals, inorganic glasses and plastics. Thesize and shape of the solid support may also vary without departing fromthe scope of the invention. A solid support may be planar, a solidsupport may be a well, i.e. a 384 well plate, or alternatively, a solidsupport may be a bead or a slide.

R⁸ may be attached to the R⁹ in a wide variety of ways, as will beappreciated by those in the art. R⁸, for example, may either besynthesized first, with subsequent attachment to the solid support, ormay be directly synthesized on the solid support. R⁹ and R⁸ may bederivatized with chemical functional groups for subsequent attachment ofthe two. For example, the solid support may be derivatized with achemical functional group including, but not limited to, amino groups,carboxyl groups, oxo groups or thiol groups. Using these functionalgroups, the R⁸ may be attached using functional groups either directlyor indirectly using linkers. Alternatively, R⁸ may also be attached tothe surface non-covalently. For example, a biotinylated R⁸ can beprepared, which may bind to surfaces covalently coated withstreptavidin, resulting in attachment. Alternatively, R⁸ may besynthesized on the surface using techniques such as photopolymerizationand photolithography. Additional methods of attaching R⁸ to a surfaceand methods of synthesizing nucleic acids on surfaces are well known inthe art, i.e. VLSIPS technology from Affymetrix (e.g., see U.S. Pat. No.6,566,495, and Rockett and Dix, “DNA arrays: technology, options andtoxicological applications,” Xenobiotica 30(2):155-177, all of which arehereby incorporated by reference in their entirety).

In each of the foregoing embodiments for molecular biosensors havingformula (III), the first nucleic acid construct, R¹—R²—R³ and the secondnucleic acid construct, R⁴—R⁵—R⁶, may optionally be attached to eachother by a linker R^(LA) to create tight binding bivalent ligands.Typically, the attachment is by covalent bond formation. Alternatively,the attachment may be by non covalent bond formation. In one embodiment,R^(LA) attaches R¹ of the first nucleic acid construct to R⁴ of thesecond nucleic acid construct to form a molecule comprising:

In a further embodiment, R^(LA) attaches R² of the first nucleic acidconstruct to R⁵ of the second nucleic acid construct to form a moleculecomprising:

In yet another embodiment, R^(LA) attaches R3 of the first nucleic acidconstruct to R7 of the second nucleic acid construct to form a moleculecomprising:

Generally speaking, R^(LA) may be a nucleotide sequence from about 10 toabout 100 nucleotides in length. The nucleotides comprising R^(LA) maybe any of the nucleotide bases in DNA or RNA (A, C, T, G in the case ofDNA, or A, C, U, G in the case of RNA). In one embodiment, R^(LA) iscomprised of DNA bases. In another embodiment, R^(LA) is comprised ofRNA bases. In yet another embodiment, R^(LA) is comprised of modifiednucleic acid bases, such as modified DNA bases or modified RNA bases.Modifications may occur at, but are not restricted to, the sugar 2′position, the C-5 position of pyrimidines, and the 8-position ofpurines. Examples of suitable modified DNA or RNA bases include2′-fluoro nucleotides, 2′-amino nucleotides, 5′-aminoallyl-2′-fluoronucleotides and phosphorothioate nucleotides (monothiophosphate anddithiophosphate). In a further embodiment, R^(LA) is comprised ofnucleotide mimics. Examples of nucleotide mimics include locked nucleicacids (LNA), peptide nucleic acids (PNA), and phosphorodiamidatemorpholine oligomers (PMO). Alternatively, R^(LA) may be a polymer ofbifunctional chemical linkers. In one embodiment the bifunctionalchemical linker is heterobifunctional. Suitable heterobifunctionalchemical linkers include sulfoSMCC(Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), andIc-SPDP(N-Succinimidyl-6-(3′-(2-PyridylDithio)-Propionamido)-hexanoate). Inanother embodiment, the bifunctional chemical linker ishomobifunctional. Suitable homobifunctional linkers includedisuccinimidyl suberate, disuccinimidyl glutarate, and disuccinimidyltartrate. An exemplary R^(LA) is the phosphoramidate form of Spacer 18comprised of polyethylene glycol. In one embodiment, R^(LA) is fromabout 1 to about 500 angstroms in length. In another embodiment, R^(LA)is from about 20 to about 400 angstroms in length. In yet anotherembodiment, R^(LA) is from about 50 to about 250 angstroms in length.

(b) Three Component Molecular Biosensors without a Solid Support

In an alternative embodiment of the three-component biosensor, thebiosensor does not comprise a solid support. For instance, in someembodiments, the three-component molecular biosensor comprises threeconstructs, which together have formula (VI):R¹—R²—R³;R⁴—R⁵—R⁶; andat least one R⁷—R⁸;  (VI)wherein:

-   -   R¹ is an epitope-binding agent that binds to a first epitope on        a target molecule;    -   R² is a flexible linker attaching R¹ to R³;    -   R³ and R⁶ are a first pair of nucleotide sequences that are        complementary to two distinct regions on R⁸;    -   R⁵ is a flexible linker attaching R⁴ to R⁶;    -   R⁶ is an epitope-binding agent that binds to a second epitope on        a target molecule;    -   R⁸ is a nucleotide construct comprising a first region that is        complementary to R³ and a second region that is complementary to        R⁶, such that when R³ and R⁶ associated with R⁸, an endonuclease        restriction site is reconstituted;    -   R⁷ is a signaling molecule.

R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ may be as defined above forthree-component molecular biosensors having formula (II). R⁸ may be asdescribed in Section (II)(a) above.

In some embodiments for molecular biosensors having Formula (VI), R⁷ maycomprise two signaling molecules, each attached to one strand of adouble-stranded nucleotide sequence comprising R⁸. Cleavage of therestriction enzyme recognition site results in the release andseparation of the two signaling molecules, resulting in a detectable andquantifiable change in signal intensity. Exemplary detections meanssuitable for use in the molecular biosensors include fluorescentresonance energy transfer (FRET), lanthamide resonance energy transfer(LRET), fluorescence cross-correlation spectroscopy, fluorescencequenching, fluorescence polarization, flow cytometry, scintillationproximity, luminescence resonance energy transfer, direct quenching,ground-state complex formation, chemiluminescence energy transfer,bioluminescence resonance energy transfer, excimer formation,colorimetric substrates detection, phosphorescence, electrochemicalchanges, and redox potential changes.

III. Methods for Utilizing a Molecular Biosensor

A further aspect of the invention encompasses the use of the molecularbiosensors of the invention in several applications. In certainembodiments, the molecular biosensors are utilized in methods fordetecting one or more target molecules. In other embodiments, themolecular biosensors may be utilized in kits and for therapeutic anddiagnostic applications.

In one embodiment, the molecular biosensors may be utilized fordetection of a target molecule. The method generally involves contactinga molecular biosensor of the invention with the target molecule. Todetect a target molecule utilizing two-component biosensors, the methodtypically involves target-molecule induced co-association of twoepitope-binding agents (present in the molecular biosensor of theinvention) that each recognize distinct epitopes on the target molecule.The epitope-binding agents each comprise complementary oligonucleotides.Co-association of the two epitope-binding agents with the targetmolecule results in annealing of the two complementary oligonucleotidessuch that a detectable signal is produced. Typically, the detectablesignal is produced by any of the detection means known in the art or asdescribed herein. Alternatively, for three-component biosensors,co-association of the two epitope-binding agent constructs with thetarget molecule results in hybridization of each signaling oligos to theoligonucleotide construct. Binding of the two signaling oligo to theoligonucleotide construct brings them into proximity such that adetectable signal is produced.

In one particular embodiment, a method for the detection of a targetmolecule that is a protein or polypeptide is provided. The methodgenerally involves detecting a polypeptide in a sample comprising thesteps of contacting a sample with a molecular biosensor of theinvention. By way of non-limiting example, the molecular biosensor maycomprise two aptamers recognizing two distinct epitopes of a protein, adouble stranded polynucleotide containing binding site for DNA bindingprotein and an aptamer recognizing a distinct epitope of the protein, anantibody and an aptamer recognizing distinct epitopes of the protein, adouble stranded polynucleotide containing a binding site for a DNAbinding protein and an antibody recognizing a distinct epitope of theprotein, two antibodies recognizing two distinct epitopes of theprotein, two double stranded polynucleotide fragments recognizing twodistinct sites of the protein, two single stranded polynucleotideelements recognizing two distinct sequence elements of another singlestranded polynucleotide.

The molecular biosensor may also detect formation of aprotein-polynucleotide complex using a double stranded polynucleotidefragment (containing the binding site of the protein) labeled with afirst signaling oligonucleotide and the protein labeled with a secondsignaling oligonucleotide (FIGS. 13 and 14). Or alternatively, thebiosensor may comprise a first polynucleotide fragment with acomplementary overhang to a second polynucleotide fragment, such that inthe presence of a DNA-binding protein, the first polynucleotide fragmentassociates with the second polynucleotide fragment to create theDNA-binding protein epitope, which allows association of the DNA-bindingprotein with the DNA-binding protein epitope. The molecular biosensormay also comprise a molecular biosensor that allows for the directdetection of the formation of a protein-protein complex using twocorresponding proteins labeled with signaling oligonucleotides.

In another embodiment, the molecular biosensors may be used to detect atarget molecule that is a macromolecular complex in a sample. In thisembodiment, the first epitope is preferably on one polypeptide and thesecond epitope is on another polypeptide, such that when amacromolecular complex is formed, the one and another polypeptides arebought into proximity, resulting in the stable interaction of the firstaptamer construct and the second aptamer construct to produce adetectable signal, as described above.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

The term “antibody” generally means a polypeptide or protein thatrecognizes and can bind to an epitope of an antigen. An antibody, asused herein, may be a complete antibody as understood in the art, i.e.,consisting of two heavy chains and two light chains, or be selected froma group comprising polyclonal antibodies, ascites, Fab fragments, Fab′fragments, monoclonal antibodies, chimeric antibodies humanizedantibodies, and a peptide comprising a hypervariable region of anantibody.

The term “aptamer” refers to a polynucleotide, generally a RNA or a DNAthat has a useful biological activity in terms of biochemical activity,molecular recognition or binding attributes. Usually, an aptamer has amolecular activity such as binding to a target molecule at a specificepitope (region). It is generally accepted that an aptamer, which isspecific in its binding to any polypeptide, may be synthesized and/oridentified by in vitro evolution methods

As used herein, “detection method” means any of several methods known inthe art to detect a molecular interaction event. The phrase “detectablesignal”, as used herein, is essentially equivalent to “detectionmethod.”

The term “epitope” refers generally to a particular region of a targetmolecule. Examples include an antigen, a hapten, a molecule, a polymer,a prion, a microbe, a cell, a peptide, polypeptide, protein, a nucleicacid, or macromolecular complex. An epitope may consist of a smallpeptide derived from a larger polypeptide. An epitope may be a two orthree-dimensional surface or surface feature of a polypeptide, proteinor macromolecular complex that comprises several non-contiguous peptidestretches or amino acid groups.

The term “epitope binding agent” refers to a substance that is capableof binding to a specific epitope of an antigen, a polypeptide, a nucleicacid, a protein or a macromolecular complex. Non-limiting examples ofepitope binding agents include aptamers, thioaptamers, double-strandedDNA sequence, peptides and polypeptides, ligands and fragments ofligands, receptors and fragments of receptors, antibodies and fragmentsof antibodies, polynucleotides, coenzymes, coregulators, allostericmolecules, peptide nucleic acids, locked nucleic acids,phosphorodiamidate morpholino oligomers (PMO) and ions. Peptide epitopebinding agents include ligand regulated peptide epitope binding agents.

The term “epitope binding agent construct” refers to a construct thatcontains an epitope-binding agent and can serve in a “molecularbiosensor” with another molecular biosensor. Preferably, an epitopebinding agent construct also contains a “linker,” and an “oligo”. Anepitope binding agent construct can also be referred to as a molecularrecognition construct.

The term “target molecule,” as used herein, refers to a molecule thatmay be detected with a biosensor of the invention. By way ofnon-limiting example, a target may be a biomolecule such as an antigen,a polypeptide, a protein, a nucleic acid, a carbohydrate, or amacromolecular complex thereof. Alternatively, a target may be a hapten,a molecule, a polymer, a prion, a microbe, a cell, or a macromolecularcomplex thereof.

The term “signaling molecule,” as used herein, refers to any substanceattachable to a polynucleotide, polypeptide, aptamer, nucleic acidcomponent, or other substrate material, in which the substance isdetectable by a detection method. Non-limiting examples of labelsapplicable to this invention include but are not limited to luminescentmolecules, chemiluminescent molecules, fluorochromes, fluorescentquenching agents, colored molecules, radioisotopes, scintillants,massive labels (for detection via mass changes), biotin, avidin,streptavidin, protein A, protein G, antibodies or fragments thereof,Grb2, polyhistidine, Ni²⁺, Flag tags, myc tags, heavy metals, enzymes,alkaline phosphatase, peroxidase, luciferase, electron donors/acceptors,acridinium esters, and colorimetric substrates. The skilled artisanwould readily recognize other useful labels that are not mentionedabove, which may be employed in the operation of the present invention.

EXAMPLES

The following examples illustrate various iterations of the invention.

Example 1. Two-Component Molecular Biosensors Comprising a SingleNicking Restriction Endonuclease Recognition Site

This example describes a method for the rapid and sensitive detection ofa target molecule using a two-component molecular biosensor. This methodis based on the target-driven association of two constructs containingepitope-binding agents that recognize two distinct epitopes of a target(FIG. 1). These two epitope-binding agent constructs each comprise asingle-stranded nucleotide sequence. Each single-stranded sequencecomprises a complementary 3′ end sequence, and a restrictionendonuclease recognition site. The complementary 3′ end sequences arebrought into close proximity when the epitope binding agentsco-associate with a molecular target, resulting in annealing of thecomplementary 3′ end sequences such that, when the complementary regionsare extended by a nucleotide polymerase, a double-stranded nucleic acidcomprising a restriction enzyme recognition site is reconstituted. Anicking restriction endonuclease enzyme that recognizes thereconstituted restriction enzyme recognition site nicks one strand ofthe newly synthesized nucleic acid duplex. A DNA polymerase extends asecond nucleic acid thereby displacing the first displaced strand, andproducing a displaced single-stranded nucleic acid. The second extendedstrand is then nicked and the extension/displacement cycle may berepeated to produce multiple copies of the displaced strand, therebyproviding a means of amplifying the signal. The produced nicked strandmay then be quantified using one of several different methods. Threepossible methods are detailed below.

Double-Stranded Nucleic Acid Stains

The displaced DNA strand may be detected by annealing with acomplementary nucleic acid sequence, to form double stranded DNA whichmay be detected using stains that specifically bind double stranded DNA(FIG. 1 E1).

Detection Using a Type IIS Endonuclease Construct

The displaced DNA strand may be detected by annealing to a type IISendonuclease construct (FIG. 1 E2). The type IIS endonuclease constructcomprises a double-stranded DNA region, and a single-stranded DNAregion. The single stranded DNA region of the construct is complementaryto the displaced DNA strand, such that when the displaced strandassociates with the construct, a type IIS endonuclease recognition siteis reconstituted. The construct also comprises a detection means, suchthat when a type IIS endonuclease cleaves the construct, the detectionmeans are released from the construct, and a detectable signal isproduced.

Detection Using a Linker Construct

The displaced strand may be detected by annealing to a linker construct(FIG. 1 E3). In general, a linker construct would comprise adouble-stranded DNA region, and a single-stranded DNA region. The linkerconstruct also comprises a detection means linked to a pair ofcomplementary oligonucleotides. The pair of complementaryoligonucleotides, and the detection means linked to them, are linked tothe double-stranded and single-stranded DNA regions through flexiblelinkers. The single stranded DNA region of the construct iscomplementary to the displaced DNA strand, such that when the displacedstrand associates with the construct, a double-stranded restrictionendonuclease recognition site is reconstituted. In the presence of arestriction endonuclease, double-stranded DNA region and the displacedstrand are cleaved at the endonuclease site resulting in the separationof the detection means, and a detectable signal is produced.

Example 2. Two Component Molecular Biosensors Comprising Two NickingRestriction Endonuclease Recognition Sites

In an alternative embodiment of the target detection method described inExample 1 above, the single-stranded nucleotide sequences of theepitope-binding agent constructs comprise two restriction enzymerecognition sites (FIG. 2). In some embodiments, the restriction sitesmay be distal to each other (FIG. 2 C1). In these embodiments, DNApolymerase extends the double-stranded nucleic acid producing twodisplaced strands. The nicking, and the extension/displacement cycle maybe repeated to produce multiple copies of the displaced strands toamplify the signal. The displaced strands produced are complementary,and may be detected using stains that specifically bind double strandedDNA (FIG. 2 C2) as described in Example 1 above.

In other embodiments the restriction endonuclease sites may be proximalto each other. In these embodiments, the displaced strands are notcomplementary to each other, but may be detected by annealing to typeIIS endonuclease constructs (FIG. 2 F1 and F2) or linker constructs(FIGS. 2 G1 and G2) as described in Example 1 above.

Example 3. Validation of Three Component Molecular Biosensor

This example describes a method for the rapid and sensitive detection ofa target molecule using a three-component molecular biosensor (FIG. 3).The three component biosensor comprises two epitope-binding agentconstructs and a single-stranded oligonucleotide construct comprising arestriction enzyme recognition site. The oligonucleotide construct isimmobilized on a solid support and comprises a signaling molecule.Detection of a target molecule typically involves target-moleculeinduced co-association of the two epitope-binding agent constructs thateach recognizes distinct epitopes on the target molecule. Theepitope-binding agent constructs each comprise a single-strandednucleotide sequence that are not complementary to each other, but arecomplementary to two distinct regions of an oligonucleotide construct.Co-association of the two epitope-binding agent constructs with thetarget molecule results in hybridization of single-stranded nucleotidesequences to distinct regions of the oligonucleotide construct. Thistripartite construct comprising the two single-stranded nucleic acidsequences and the oligonucleotide construct reconstitutes a restrictionendonuclease recognition site. When a restriction endonuclease cleavesthe restriction endonuclease site, releasing the signaling molecule fromthe solid support for measurement.

To validate the assay described, epitope binding agent constructs wereincubated with 0, 10, 20 and 30 nM concentrations of target molecule inthe presence of an oligonucleotide construct in a master mix containingthe restriction enzyme HincII. The reaction was then loaded onto anagarose gel, and the products of the restriction digestion reactionresolved. The results show that in the absence of target molecule, only20% of the oligonucleotide construct was digested by the HincII enzyme.Adding increasing concentrations of the target molecule resulted inincreasing digestion of the oligonucleotide construct (FIG. 4).

Example 4. Three Component Molecular Biosensor Immobilized on MagneticBeads

In this example, the oligonucleotide construct described in Example 3was labeled with FAM, then conjugated with biotin and immobilized onstreptavidin magnetic beads (SMB). The oligonucleotide construct wasincubated with pre-equilibrated SMB in 50 mM Tris, 150 mM NaCL, 0.02%tween-20, pH 8.0 at room temperature for 50 minutes. The beads were thenwashed three times. Master mix (2 μl) was added into each tube, andother components were added as detailed in Table 1 below. The finalvolume of the reaction was 20 μl/tube in 1× reaction buffer (20 mM Tris,100 mM NaCl, 2 mM MgCl₂, 0.2 mM DTT, 0.2 mg/ml BSA) and HincII. Thereaction was incubated at room temperature for 35 minutes, and 10 μl ofthe reaction was then transferred into a 384-well plate and read at ex.485 nm, em. 535 nm (FIG. 5).

A similar experiment was performed using an oligonucleotide constructlabeled with horse radish peroxidase (HRP). Master mix (2 μl) was addedinto each tube, and other components were added as detailed in Table 1below. The final volume of the reaction was 35 μl/tube in 1× reactionbuffer (20 mM Tris, 100 mM NaCl, 2 mM MgCl2, 0.2 mg/ml BSA) and HincII.The reaction was incubated at room temperature for 40 minutes, and 30 μlof the reaction was then transferred into a 96-well plate and mixed with40 μl chemiluminescent ELISA substrate, and luminescence read (FIG. 6).

Example 5. Three Component Molecular Biosensor Immobilized on MagneticBeads and Sequential Addition of Target and Restriction Enzyme

In a variation of the above conditions, the FAM-labeled oligonucleotideconstruct immobilized on beads was mixed with the epitope bindingconstructs and the target molecule, and the mixture incubated at RT inbinding buffer (50 mM Tris, pH 8.0, 150 mM NaCl₂, 0.02% Tween-20, 0.2mg/ml BSA) for 20 min, then washed 1× with 50 μl binding buffer. Thiswas followed by the addition of 1×HincII buffer (20 mM Tris, pH 8.0, 100mM NaCl, 2 mM MgCl₂, 0.2 mM DTT, 0.2 mg/ml BSA) with HincII, for a finalvolume of 25 μl. The mixture was incubated at room temperature for 50min. HincII-mediated release of FAM signal was measured using 22 μl ofthe reaction in a 384 well plate (FIG. 7).

Example 6. Three Component Molecular Biosensor Immobilized on PlateSurface

In this Example, a FAM or HRP-labeled oligonucleotide constructdescribed in Example 3 was immobilized on a plate (FIG. 8). The platewas coated with 30 μl of 400 nM streptavidin and incubated overnight at4° C. The plate was then blocked with 1% BSA at room temperature for 3hr, and washed with TBS 3 times. This was followed by the addition of 30μl 200 nM S4, 180 nM S3, 160 nM A2-FAM, and incubated at roomtemperature for 2.5 hr, then washed with TBS 4 times. 25 μl of eachsample was added, followed by 1×HincII buffer (20 mM Tris, pH 8.0, 100mM NaCl, 2 mM MgCl₂, 0.2 mM DTT, 0.2 mg/ml BSA) and 3 units of HincIIenzyme. The reaction was incubated at room temperature for 30 min. ForFAM, 20 μl of the reaction was taken into a 384-well plate and read atex. 485 nm, em. 535 nm (FIG. 9A). For HRP, 20 μl was taken into an ELISAplate, 20 μl of TMB/H₂O₂ mix was added and the OD450 nm was measured(FIG. 9B)

Example 7. Three Component Molecular Biosensor Comprising SignalingOligonucleotide Construct with Double-Stranded Nucleotide Regions

This Example describes a method for the rapid and sensitive detection ofa target molecule using a three-component molecular biosensor (FIG. 10).The three component biosensor comprises two epitope-binding agentconstructs and an oligonucleotide construct comprising regions that aredouble-stranded and regions that are single-stranded. Theoligonucleotide construct also comprises two signaling molecules, eachattached to one strand of the double-stranded region of theoligonucleotide construct. Detection of a target molecule typicallyinvolves target-molecule induced co-association of the twoepitope-binding agent constructs that each recognize distinct epitopeson the target molecule. The epitope-binding agent constructs eachcomprise non-complementary single-stranded nucleotide sequences that arecomplementary to two distinct, but contiguous single-stranded regions ofthe oligonucleotide construct, producing a double-stranded nucleic acidcomprising a restriction enzyme recognition site. A type IIS restrictionendonuclease enzyme releases the signaling molecule from the doublestranded nucleic acid, resulting in a detectable and quantifiable changein signal intensity.

The oligonucleotide construct, the epitope-binding constructs, and therestriction enzyme BcgI were incubated in the presence or absence ofmolecular target in buffer (100 mM NaCl, 50 mM Tris, pH 7.9, 2 mM MgCl₂,0.2 mM DTT, 0.2 mg/ml BSA, 20 μM SAM) in a final reaction volume of 20μl. The reaction mixture was incubated at room temperature. Samples weretaken at time 0 and every 10 minutes for measurement of FAM fluorescence(Table 1 and FIG. 11).

TABLE 1 Signaling oligonucleotide construct 60 nM Epiptopeoiligonucleotide constrct 1 20 nM Epiptope oiligonucleotide constrct 120 nM Molecular target 0 20 nM Bcgl 2 units 2 units  0 min 0 0 10 min125 393 20 min 345 888 30 min 643 1417 40 min 689 1833 50 min 925 230860 min 1086 2594 70 min 1208 2839 80 min 1210 3017 90 min 1508 3321 100min  1524 3295

Example 8. Three Component Molecular Biosensor Comprising SignalingOligonucleotide Construct with Double-Stranded Nucleotide Regions, withAmplified Signal

This Example describes a three-component molecular biosensor wherein thethree component biosensor comprises two epitope-binding agent constructsand an oligonucleotide construct comprising regions that aredouble-stranded and regions that are single-stranded. Theoligonucleotide construct also comprises two signaling molecules, eachattached to one strand of the double-stranded region of theoligonucleotide construct. The single-stranded regions of theoligonucleotide construct of this example are not contiguous, such thatthe signaling oligonucleotide construct comprises alternatingdouble-stranded and single stranded regions (FIG. 12). Detection of atarget molecule typically involves target-molecule inducedco-association of two epitope-binding agent constructs that eachrecognize distinct epitopes on the target molecule. The epitope-bindingagent constructs each comprise non-complementary single-strandednucleotide sequences that are complementary to two distinctnon-contiguous regions of the oligonucleotide construct. Co-associationof the two epitope-binding agent constructs with the target moleculeresults in annealing of each signaling oligonucleotide to theoligonucleotide construct, producing a double-stranded nucleic acidcomprising a restriction enzyme recognition site. A type IIS restrictionendonuclease enzyme releases the signaling molecule from the doublestranded nucleic acid, resulting in a detectable and quantifiable changein signal intensity. The restriction endonuclease enzyme also cleaves onthe other side of the recognition sequence, within the double-strandedregion of the signaling oligo construct resulting in the dissociation ofthe complex comprising the target and the epitope binding constructs.The complex is now free to associate with a new signalingoligonucleotide construct resulting in amplification of the signalgenerated from a single target.

What is claimed is:
 1. A molecular biosensor comprising two constructs,the constructs comprising:R¹—R²—R³; andR⁴—R⁵—R⁶;  (I) wherein: R¹ is an epitope-binding agent that binds to afirst epitope on a target molecule; R² is a flexible linker attaching R¹to R³; R³ is a single stranded nucleotide sequence comprising R⁷ and R⁸;R⁷ is a nucleotide sequence comprising at least one nicking restrictionendonuclease recognition site; R⁸ is a nucleotide sequence complementaryto R⁹; R⁶ is a single stranded nucleotide sequence comprising R⁹; R⁹ isa nucleotide sequence complementary to R⁸, such that when R⁸ and R⁹associate to form an annealed complex in presence of a polymerase, R⁸and R⁹ are extended by the polymerase to form a nucleotide sequencecomplementary to R⁷, forming at least one double-stranded nickingrestriction endonuclease recognition site; R⁵ is a flexible linkerattaching R⁴ to R⁶; R⁴ is an epitope-binding agent that binds to asecond epitope on the target molecule.
 2. The molecular biosensor ofclaim 1, wherein the free energy for association of R⁸ and R⁹ are fromabout −5.5 kcal/mole to about −8.0 kcal/mole at a temperature from about21° C. to about 40° C., and a salt concentration from about 1 mM toabout 100 mM.
 3. The molecular biosensor of claim 1, wherein R³ and R⁶are independently from about 2 to about 40 nucleotides in length.
 4. Themolecular biosensor of claim 1, wherein R³ comprises at least onenicking restriction endonuclease recognition site.
 5. The molecularbiosensor of claim 1, wherein R³ comprises at least two nickingrestriction endonuclease recognition sites.
 6. The molecular biosensorof claim 5, wherein R³ comprises at least two nicking restrictionendonuclease recognition sites distal to each other.
 7. The molecularbiosensor of claim 5, wherein R³ comprises at least two nickingrestriction endonuclease recognition sites proximal to each other. 8.The molecular biosensor of claim 1, wherein R⁶ comprises at least onenicking restriction endonuclease recognition site.
 9. The molecularbiosensor of claim 1, wherein R⁶ comprises at least two nickingrestriction endonuclease recognition sites.
 10. The molecular biosensorof claim 9, wherein R⁶ comprises at least two nicking restrictionendonuclease recognition sites distal to each other.
 11. The molecularbiosensor of claim 9, wherein R⁶ comprises at least two nickingrestriction endonuclease recognition sites proximal to each other.
 12. Amethod for determining presence of a target molecule in a sample, themethod comprising: a) combining a molecular biosensor of claim 1 withthe target molecule; b) extending R⁸ and R⁹ to form a nucleotidesequence complementary to R⁷; c) contacting the molecular biosensor witha nicking restriction endonuclease that recognizes R⁷; d) repeatingsteps b and c to amplify the displaced single-stranded nucleotidesequence; e) measuring the release of the displaced single-strandednucleotide sequence, wherein an increase in signal indicates thepresence of the target molecule.